WO2001094601A2 - Hero resistance gene conferring resistance to nematodes - Google Patents

Abstract

The invention relates to a hero resistance gene and gene fragments thereof, for the biosynthesis of proteins which generate resistance to nematodes, especially Globodera cyst nematodes; to their isolation and to their use for the genetic transformation of susceptible plants such as potato, tomato and aubergine. It was found that the hero resistance gene codes the protein sequence according to figure 1. The corresponding gene sequence was used for producing plants with resistance to Globodera cyst nematodes using gene technology.

Description

-flTero resistance gene against nematodes

description

The invention relates to a hero resistance gene and gene fragments thereof for the biosynthesis of proteins which bring about resistance to nematodes, in particular to globodera cyst nematodes, their isolation and their use for the genetic engineering transformation of susceptible plants, such as e.g. Potato, tomato and eggplant. The field of application is in particular agriculture.

Nematodes (nematodes) are animal pests that cause great problems in agriculture because they are difficult to control and chemical control requires the use of highly toxic substances. On the other hand, permanent stages of the nematodes can survive in the soil for up to 10 years, making the cultivation of susceptible (susceptible) plants on infested fields impossible without chemical control with nematicides for a long time.

The cyst nematodes Globodera rostochiensis and Globodera pallida cause great damage when growing nightshade plants in the temperate climate zone (e.g. Central Europe). Of these Globodera species, the potato (Solarium tuberosum), but also the tomato (Lycopersicon esculentum) and the aubergine (Solarium melongena), as well as other plant species, are particularly affected. Particularly when growing potatoes, significant crop losses can occur due to infestation with cyst nematodes.

Five pathotypes (Rol to Ro5) have now been described for Globodera rostochiensis, which can be differentiated by corresponding resistance genes occurring in cultivars and wild material (gene for gene interaction, Flor 1956, Adv. Genet. 8: 29-54). 3 pathotypes are known for Globodera pallida (Pal to Pa3).

Genetic mapping with the help of molecular markers made it possible to locate the hero resistance gene on chromosome 4 of the tomato in 1995 (Ganal et al. 1995; Mol. Plant Micr. Interact. 6: 886-891). Genetically closely linked markers could be identified. In continuation of the work published in 1995, it became known that genes with homology to known resistance genes from the NBS-LRR class could be found in the region. Among other things, the deduced amino acid sequence of the marker HR1 (Ganal et al. 1995) shows significant homology to the amino acid sequence of the Prf gene from the tomato, which plays a role in the interaction with Pseudomonas syringae (Salemeron et al., 1996; Cell 86: 123-133). The invention was based on the object of precisely localizing the molecular resistance to chromosome 4 of the tomato, isolating it molecularly and sequencing it, and its use for genetic engineering transformation in susceptible plants, such as tomato, potato, eggplant, to form a nematode To create resistance.

The invention is based on our own findings that the nematode resistance gene hero from the tomato brings about complete resistance to all pathotypes of the cyst nematode Globodera rostochiensis and probably also partial resistance to Globodera pallida. It therefore has an extremely broad spectrum of resistance to cyst nematodes. A detailed description of the hero gene was first given in 1971 by Ellis and Maxon-Smith 1971 (Euphytica 20: 93-101).

The object of the invention was achieved with gene A, the coding region of which is sequence 2 with 3909 bp (cf. sequence listing) and which could be isolated from a large number of candidate genes from the gene family of the NBS-LRR resistance gene homologues. The hero resistance gene was found to encode the protein sequence of Figure 1.

The invention relates to the hero resistance gene with the gene sequence A, which comprises bp 16642 to 21002 of FIG. 4 (cf. also sequence 1 in the sequence listing), the nucleotides 16666 to 16759 and 20574 to 20931 being two non-coding introns. The invention also relates to a gene with sequence 2, which comprises 3909 bp and has the coding sequence of gene A. The invention also relates to a protein with the amino acid sequence shown in FIG. 1, and to derivatives of the genes which code for proteins in which some of the amino acids from the amino acid sequence are missing or are substituted by others or in which amino acids have been added, but which contain the have the same properties. The present invention further relates to vectors and plasmids which contain the hero gene.

The inventive achievement with regard to the isolation of the hero resistance gene lies in particular in isolating from these resistance gene analogues, despite their great similarity, the gene which causes resistance to the individual pathotypes of Globoderea rostochiensis. Due to the up to 98% identical nucleic acid sequence, the genes could only be distinguished after the entire region had been sequenced. Cross analyzes and complementation of susceptible plants by the transformation with DNA segments which contained one or more genes of the gene family finally led to the identification of the hero gene which codes for a protein of the sequence of FIG. 1. The following applications are possible with the invention:

1. Generation of resistant or increasingly resistant plants to the nematodes Globodera rostochiensis and Globodera pallida in all plant species which are originally susceptible to these nematodes, preferably potato, tomato and eggplant, by transformation with the isolated gene using the own or third-party promoters.

The invention therefore also relates to cells and transgenic plants which have been transformed by means of vectors and plasmids.

2. Genetic engineering of the gene by inserting, adding or removing single or multiple nucleotides to create new resistance specificities to these nematodes or to create a generally improved resistance to all races of these and other nematodes.

The invention is explained in more detail below by means of the exemplary embodiments: General molecular biological techniques have been carried out according to the prior art. Enzymatic reactions were carried out according to the recommendations of the manufacturers. Most of the techniques used are described in the Molecular Cloning and Genome Analysis manuals (Sambrook et al. Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 1989; Green et al Genome Analysis - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 1997).

Exemplary embodiments and descriptions

1. Characterization of the gene family of the NBS-LRR resistance gene homologues

To characterize the gene family, the HR1 probe phage cDNA libraries of the tomato (made from root RNA of the Mogeor or VFN8 varieties) were sifted using standard methods at a hybridization temperature of 65 ° C. The washing conditions of the hybridizations were 0.5 x SSC, 0.1% SDS at 65 ° C. The identified clones were examined for their size and sequenced. Specific hybridization samples from the 5 'and 3' region of the longest clone (Wgt23) were used in addition to the HRl probe to examine genomic DNA. Genomic Southern analyzes (Figure 2) show that the hybridization probes (here HR1) detect a whole series of DNA fragments under standard conditions (65 ° C hybridization temperature) be (at least 8 to 10). Genetic mapping of these fragments on a tomato population (Tanksley et al, 1992; Genetics 132: 1141-1160) shows that many but not all of these fragments in the region map to chromosome 4 of the tomato (marked with Hero in Figure 2) which contains the hero gene. Due to the different intensities of the fragments, it is clear that some of the hybridizing fragments detect more than one gene and therefore the gene cannot be easily isolated because there is a larger gene family.

2. Isolation of the genes from the Ηero region on chromosome 4 of the tomato

In a first step, a genomic library in the binary cosmid p04541 (Jones et al. 1992; Transgenic Research 1, 285-297) was produced for the molecular isolation of the genes of resistance genomics on chromosome 4. DNA from leaves of the LA1792 (Ηero) line was extracted using a modified CTAB method (Bernatzky and Tanksley, 1986; Genetics 112: 887-898). The genomic DNA was partially digested with Sau3A and fractionated using a sucrose gradient centrifugation. DNA in the size range between 9 and 23 kb was ligated into the Rα ΗI-cut vector. After packaging the Cosmide with the Gigapacklll XL Packaging Extract (Stratagene, La Jolla, CA, USA), the resulting phages were introduced into the Escherichia coli strain XLl-Blue MRF'Kan (Stratagene). 136 bacterial pools with 1000-2000 different bacterial clones each were obtained (corresponds to approximately 4.4 genome equivalents). After DNA extraction from the individual pools, these DNA pools were sighted using primers for the ΗR1 sequence and 5 'and 3' sequences from the cDNA clones (eg Wgt23). The cosmid clones obtained were characterized according to standard methods by digestion with restriction enzymes and via hybridization with the ΗR1 probe and fragments from Wgt23. The ends of the cosmid were also sequenced. Starting from the end sequences of the isolated cosmids, further primers were derived in order to isolate additional cosmids. Because of the almost identical sequences of the gene family detected by ΗR1, it was only possible with great difficulty to assemble the gene family in a completely overlapping contig. The generation of specific probes was only possible by sequencing all cosmids and identifying regions which are specific for individual cosmids by means of sequence comparisons. Analysis of a 158 kb DNA sequence showed that 14 very similar genes are located in the Contig. Of these genes, six genes are not functional due to raster mutations, insertions or deletions. Eight genes show an intact reader and are therefore potentially functional genes. A representation of the entire contig is shown in Figure 3. 3. Identification of hero candidates from the gene family using high-resolution genetic mapping

The large number of candidate genes for hero and the known problems with large and gene duplication-containing cosmids in Agrobacterium tumefaciens required further selection of the candidates for complementation. This was done by analyzing a large F2 population from the crossing Hero (LA1792) x Ailsa Craig. 747 plants were examined by means of RFLP analysis for the interval HR1 / H293 (Ganal et al. 1995) with these two markers. For the interval CT229 / HR1, a further 207 plants with the CT229 and HRl markers were examined. All recombinant plants were self-grown and in the F3 generation (10 to 15 plants per recombinant in each case) those progeny were identified with the corresponding markers that were homozygous for the recombination event. These plants were again self-grown and the corresponding offspring could be tested in greater numbers for their nematode resistance, since they are all uniformly homozygous for the recombination event. The resistance tests were carried out according to the descriptions by Behringer (Bayer. Landw. Jahrbuch 62, SH3, 1985) or Ganal et al. (Mol. Plant Micr. Interact. 6, 886-891, 1995). The data from the resistance tests with the homozygous recombinant plants are more reliable than the normal F3 progeny analysis (Ganal et al. 1995) and show that, in contrast to the data published there, the hero gene lies between the RFLP markers ΗR1 and CT229. The exact analysis of the recombination events identified with the help of specific PCR markers from the entire region of the gene family described above shows that the hero resistance gene with the PCR markers A10E13P1 / P2 (5'GGA TGA CTT TTA TCT CAG G3 '/ 5' GGA TAG GAT AGA AAT CCA TC3 '), F1231UP1 / UP2 (5'CGT ATA TAT TTT TTC ATC TGC3V5'GAG TAT TTC ACT ATG GTG3') and F51ES25P1 / P2 (5'AAT TCA AGT CAA GGT GGA G3 '/ 5' GGA GAA AAT GAT TTG ATA TAA C3 ') can be limited to a region of approximately 60 kb. This region contains only three functional genes (genes A, B and C) from the gene family described (Figure 3). A further analysis of the recombinant plants for the range between ΗR1 and CT229 with all pathotypes (Rol to Ro5) showed that the resistance to all pathotypes is cosegregated and is therefore very likely controlled by a single gene.

4. Sequence analysis of the cosegregating region

FIG. 4 shows a region of 23.67 kb from the region coregulating with the hero gene, which contains the complete sequence of the three genes A, B and C. The DNA Sequence was determined using standard methods from the Cosmide A3-2, F12-31 and C6-25 and subclones generated therefrom. The genomic sequences of the individual genes are defined by the following sections:

Coding sequence gene A nucleotide 16642 to 21002 Coding sequence gene B nucleotide 7973 to 12130 Coding sequence gene C nucleotide 1690 to 5446

5. Transformation of susceptible tomato lines with the hero gene and complementation to resistance

To determine which of the three genes (A, B, C) is responsible for resistance to Globodera rostochiensis, three different cosmids, which contain corresponding sequence segments, were introduced into the susceptible tomato varieties Moneymaker and Ailsa Craig by means of Agrobacterium-mediated transformation. The procedure of Ling et al (Plant Cell Rep. 17: 643-847) was used for this. The three cosmids used contain the following genes:

Cosmid A3-2: nucleotide 9986 to 23670

Gen A complete, Gen B incomplete, Gen C not

Cosmid F12-31: nucleotide 6640-23671

Gen A complete, Gen B complete, Gen C not

Cosmid C6-25: nucleotide 2379-16896

Gen A partially, Gen B complete, Gen C partially

Transformed plants (T0) were confirmed using «pt-specific primers via PCR. For the resistance tests, transformed plants were self-grown and the resulting seeds were used for the resistance tests. The resistance tests with the pathotypes Rol, Ro3 and Ro5 were carried out as described (Ganal et al. 1995) at the Scottish Crop Research Institute by Dr. Amar Kumar and Dr. Mark Phillips performed. The results of the resistance tests are shown in Figure 5. Cosmid A3-2 and Cosmid F12-31 are able to transfer resistance to the originally susceptible lines Moneymaker and Ailsa Craig. The two cosmids overlap in a region of 7 kb which contains the complete gene A. This identifies gene A as the hero resistance gene. 6. Description of the gene structure of the hero gene:

The amino acid sequence (1302 amino acids) for the hero resistance gene (gene A) is shown in Figure 6. The gene contains an intron in the 5 'untranslated region (nucleotide 15292 to 16622 and two introns within the coding sequence (nucleotides 16666 to 16759 and 20574 to 20931, see also Figure 4). The calculated molecular weight is 150021 daltons. A sequence comparison with The published nematide resistance genes Mi and Gpa2 show an identity of 35% to the Mi gene and 24% to Gpal. The hero gene contains a typical P-loop (GMPGNGK), a kinase 2 (KRYLIVLDDV) and kinase 3a (GΝRVILTSR) region The C-terminal part contains 13 imperfect leucine-rich repeating units (LRR). Atypical in comparison to other resistance genes is a so-called acid coiled coil domain within the LRRs (amino acids 1025 to 1050), which is encoded by a microsatellite-like sequence Length of this microsatellite sequence represents an important distinguishing feature between the individual members of the gene family and could be based on the formation of the Sp specificity towards the nematodes.

Legends for the illustrations

Figure 1: Amino acid sequence of the hero resistance gene

Figure 2: Southern analysis with HRl. Genomic DNA from descendants of the L. pennellii mapping population (Tanksley et al., 1992) was digested with EcoRV and separated on a 1% agarose gel. After blotting on Hybond N +, the HR1 probe was hybridized at 65 ° C. Fragments that show a cosegregation with the hero locus are marked with Ηero. Other hybridizing fragments are labeled with the assigned chromosome. C4: chromosome 4, C6: chromosome 6, C3 / C12: chromosome 3 or 12; P: E. pennelli, Ε: E. esculentum

Figure 3: Genomic organization of the 14 resistance gene-like sequences at the Hero Locus. The 17 cosmid clones covering the entire region are shown. Arrows above the map show the position, relative length and direction of transcription of each open reading frame. Letters identify each individual member of the hero gene family. Cosmide A3-2 and F12-31 complement the susceptible variety Moneymaker. X indicates the approximate position of the recombination events that were used to limit the hero gene.

Figure 4: Region of 23.67 kb from the region co-segregating with the hero gene, which contains the complete sequence of the three genes A, B and C.

Figure 5: Complementation of the susceptible tomato variety Moneymaker with the cosmids A3 -2 and F 12-31. 10 to 15 plants from an FI progeny of the primary transformants were infected with G. rostochiensis 2 weeks after sowing. The cysts formed were counted after six weeks. Untransformed plants of the Moneymaker variety were infected as a control. Plants with fewer than 8 cysts are clearly resistant. Susceptible plants usually have more than 25 cysts. The presence of the gene and part of the vector was investigated by PCR with specific primers for gene A and by Southern hybridization for the 35S promoter of the T-DNA. R: resistant; s: susceptible; +: gene-specific PCR product or RFLP fragment detected; -: No PCR product or RFLP fragment detected.

Figure 6: Structure and amino acid sequence of the hero gene. The amino acid sequences for the kinase-la, kinase-2 and kinase-3 motif of the nucleotide binding site (NBS) are marked. The hydrophobic region and the leucine-rich repeating units (LRR) are also shown.

Claims

claims: 1. DNA sequences for a hero gene which code for proteins which bring about resistance to Globodera cyst nematodes. 2. Genomic DNA sequence according to spoke 1, characterized by nucleotides 16642 to 21002 (including introns) of Figure 4 (sequence 1), and resistance-mediating fragments and variants of this sequence. 3. DNA sequence according to claim 1 characterized by the sequence 2, comprising 3909 bp, and fragments or derivatives thereof, which code for proteins with the same properties. 4. Nucleic acid sequences according to one of claims 1 to 3, characterized in that they are completely complementary. 5. Recombinant vectors, comprising at least one sequence according to one of claims 1 to 4, for the transformation of plants, preferably tomato, potato and eggplant plants. 6. Recombinant plasmids comprising at least one sequence according to one of claims 1 to 4 for the transformation of plants, preferably tomato, potato and eggplant plants. 7. Use of at least one nucleotide sequence or a gene sequence (cDNA) according to one of claims 1 to 4 for the genetic engineering of plants by transformation which are completely or partially resistant to Globodera rostochiensis and / or Globodera pallida. 8. Transgenic plants and seeds, in particular tomato, potato and eggplant plants, containing the hero gene or resistance-imparting fragments and variants of this sequence. 9. Use of the hero gene according to spoke 2 by means of targeted mutagenesis for genetically engineering new resistance specificities against individual or all pathotypes of Globodera rostochiensis and / or Globodera pallida. 10. Protein with the sequence of Figure 1 (sequence 3), which causes resistance to Globodera cyst nematodes, and fragments and derivatives thereof, which have the same properties. 11. Protein with sequence 3.